Structures of the ON and OFF states of the membraneous fumarate/Citrate sensor kinases DcuS from E. coli and CitA from Geobacillus thermodenitrifcans

传感器激酶（sensorkinases，SK）是细菌对细胞外刺激作出反应的主要机制，其功能包括信号感知、跨膜信号传导和信号传递。SK的结构域组成和跨膜信号传导显示出广泛的变化。该提案旨在解决DcuS/CitA家族（富马酸盐和柠檬酸盐传感）的SK在ON和OFF状态下的结构，并了解跨膜和细胞内信号传导中结构域的重组。DcuS/CitA包含一个跨膜信号传导SK的古结构域组成。从大量的TM传感器激酶中，最近已经以细胞溶质GAF和激酶结构域耗尽的形式解决了NarQ硝酸SK的结构。NarQ代表一种独特的SK，其结构域组成与DcuS/CitA大不相同，并且很可能代表不同的信号转导模式。DcuS的E.大肠杆菌和嗜热土芽孢杆菌的密切相关的CitA是同源二聚体，各自由用于富马酸盐或柠檬酸盐结合的胞质外PASP（Per ARNT Sim）结构域、两个TM螺旋（TM 1、TM 2）、胞质PASC和激酶结构域组成。根据分析系统的蛋白质可用性，使用DcuS或CitA进行补充研究。PASP和PASC的结构已在ON和OFF状态下在隔离和膜整合结构中得到解决。结构、遗传和生物化学研究表明，PASP在底物结合时的压实触发了负责跨膜信号传导的TM 2的活塞样运动。PASC响应于TM 2在α-螺旋二聚化位点处的结构重排中的移位。这项工作是高分辨率分析和跨膜信号转导过程中的结构重排的综合观点的基础。工作将集中在（i）在TM区域的结构重排与TM 2的活塞样运动，可能与TM 2的剪刀运动相结合，（ii）在PASc的结构重排在信号传输和控制的激酶结构域。为了获得完整视图和高分辨率，将应用结构（NMR和结晶; AG Griesinger）、生物化学、遗传（AG Unden）和生物物理（AG Hinderberger，Griesinger）方法。准备工作建立了所有的结构和方法的研究，我们推测，DcuS/CitA代表的极少数SK之一，其跨膜和胞质信号是在高结构和功能分辨率的组合方法从结构生物学，生物化学和生物物理学。

Sensor kinases (SK) represent a major device of bacteria for responding to extracellular stimuli, resulting in autophosphorylation of the SK. Their function includes signal perception, transmembrane (TM) signaling, and signal transmission to the kinase. Domain composition and transmembrane signaling of the SK show broad variation. The proposal aims at solving the structure of a SK of the DcuS/CitA family (fumarate and citrate sensing) in the ON and the OFF state, and to understand restructuring of the domains in transmembrane and intracellular signaling. DcuS/CitA contain an archaetypical domain composition for transmembrane signaling SK. From the vast number of TM sensor kinases, the structure of the NarQ nitrate SK has been solved in a form depleted of the cytosolic GAF and kinase domains recently. NarQ represents a unique SK with a domain composition largely different from DcuS/CitA and stands very likely for a different mode of signal transduction. DcuS of E. coli and the closely related CitA of the thermophilic Geobacillus thermodenitrificans are homodimers each, composed of an extracytoplasmic PASP (Per ARNT Sim) domain for fumarate or citrate binding, two TM helices (TM1, TM2), a cytoplasmic PASC and the kinase domain. Studies are performed with DcuS or CitA complementarily according to availability of the proteins for the analytic systems. Structures of PASP and PASC have been solved in isolated and membrane-integral constructs in the ON and OFF states. Structural, genetic and biochemical studies show a compaction of PASP upon substrate binding that triggers a piston-like movement of TM2 which is responsible for transmembrane signaling. PASC responds to the shift of TM2 in a structural rearrangement at an alpha-helical dimerization site. This work is the basis for high resolution analysis and an integrated view of the structural rearrangements in the domains during transmembrane signaling. Work will concentrate on (i) on the structural rearrangements in the TM region with the piston-like movement of TM2 that may be combined with a scissors movement of TM2, (ii) the structural rearrangement in PASc during signal transmission and control of the kinase domain. For a complete view and high resolution, structural (NMR and crystallization; AG Griesinger), biochemical, genetic (AG Unden) and biophysical (AG Hinderberger, Griesinger) methods will be applied. Preparatory work established all constructs and the methods for the study, and we presume that DcuS/CitA represent one of the very few SK whose transmembrane and cytoplasmic signaling is within reach at high structural and functional resolution by a combined approach from structural biology, biochemistry and biophysics.